【摘要】 目的对铜绿假单胞菌所产blaVIM-2进行基因重组表达及纯化。方法以产blaVIM-2铜绿假单胞菌总基因组DNA为模板,PCR扩增blaVIM-2,将其克隆入pUCm-T载体后测定该核苷酸序列,再将blaVIM-2克隆入表达载体pET-41b(+),然后在大肠埃希菌BL21(DE3)中表达,表达产物再过Ni-NTA柱纯化。结果PCR扩增出大小为801bp的基因片段,与GenBank上同类酶的基因序列同源性为100%。此基因能在大肠埃希菌中大量表达,蛋白分子质量大约为56000u。结论对VIM-2金属酶的成功表达及纯化为进一步做酶动力学及酶的其他分子生物学特性研究奠定了基础。更多还原

【Abstract】 Objective To carry out sequence analysis, prokaryotic expression and purification of VIM-2 metallo-β-lactamase from Pseudomonas aeruginosa. Methods The bla_ VIM-2 was amplified by PCR and sequenced after being subcloned into pUCm-T vector. The bla_ VIM-2 was cloned into pET-41b (+) vector, and expressed in E. coli BL21 (DE3), and then purified with Ni-NTA. Results Sequence of bla_ VIM-2 shared 100% amino acid identity with bla_ VIM-2 that already registered in GenBank. The bla_ VIM-2 can be expressed in E. coli BL21 (DE3), purified with Ni-NTA. Its molecular weight is about 56 000 u. Conclusions The successful prokaryotic expression and purification of bla_ VIM-2 lays a good foundation for enzymological and molecular biological study of VIM-2 metallo-β-lactamase.更多还原